The 300kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46kDa cation- dependent MPR (CD-MPR) play a key role in lysosome biogenesis by delivering ~60 different newly synthesized acid hydrolases to the lysosome by binding to mannose 6-phosphate (M6P) residues on their N-glycans. Disruption of this essential targeting pathway results in the most severe of the human lysosomal storage disorders, mucolipidosis II. In addition to lysosomal enzymes, the repertoire of identified extracellular ligands of the CI-MPR includes a diverse spectrum of M6P-containing proteins, such as growth factors (e.g., transforming growth factor-2) and pathogenic viruses (e.g., herpes simplex, varicella-zoster), and their interaction with the MPRs can result in the growth factor's activation or degradation, and facilitate viral entry into mammalian cells. The multifunctional CI-MPR also binds the non-M6P-containing ligands plasminogen, urokinase-type plasminogen activator receptor (uPAR), insulin-like growth factor-II (IGF-II), and retinoic acid. Together, these unique binding properties of the CI-MPR mediate its ability to regulate cell growth and motility, and to function as a tumor suppressor. However, the molecular basis governing the intracellular trafficking and ligand binding properties of these receptors has not been fully defined. Recent studies from our laboratory have provided the first, and to date the only, structural views of the CD-MPR's extracellular domain, and two out of the three carbohydrate binding sites of the CI-MPR. In the current proposal, we will use crystallographic and NMR approaches to determine the structure of the MPRs' carbohydrate (Aims 1 & 3) and plasminogen (Aim 2) binding sites under conditions which are physiologically relevant, including acidic conditions which are key for the ability of the receptors to release their cargo to undergo multiple rounds of lysosomal enzyme delivery. Phosphodiester-containing proteins will be identified using domain 5 of the CI-MPR as a novel affinity probe (Aim 1). Crystallographic and solution structures of the MPRs' cytoplasmic domain in the absence and presence of adaptor proteins will be determined (Aim 4). These studies will be complemented by EPR spectroscopic analyses of MPRs containing site-specific spin labels (Aim 4). The long term goal is to understand the molecular mechanisms by which these essential receptors carry out their diverse biological functions. These studies will also provide the structural basis for the design of improved therapeutics for the treatment of lysosomal storage disorders, and novel inhibitors of viral infection and growth factor activation.